Phosphorylation-activity relationships of AMPK and acetyl-CoA carboxylase in muscle
01 Jun 2002-Journal of Applied Physiology (American Physiological Society)-Vol. 92, Iss: 6, pp 2475-2482
TL;DR: Data provide the first evidence of a direct link between extent of phosphorylation of these proteins at sites recognized by the antibodies and activity of the enzymes in electrically stimulated muscle and in muscle of rats running on the treadmill.
Abstract: AMP-activated protein kinase (AMPK) is activated during muscle contraction in response to the increase in AMP and decrease in phosphocreatine (PCr). Once activated, AMPK has been proposed to phosph...
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TL;DR: It is shown that polyphenols, including resveratrol and the synthetic polyphenol S17834, increase SIRT1 deacetylase activity, LKB1 phosphorylation at Ser428, and AMPK activity, which suggests that Sirt1 functions as a novel upstream regulator for L KB1/AMPK signaling and plays an essential role in the regulation of hepatocyte lipid metabolism.
Abstract: Resveratrol may protect against metabolic disease through activating SIRT1 deacetylase. Because we have recently defined AMPK activation as a key mechanism for the beneficial effects of polyphenols on hepatic lipid accumulation, hyperlipidemia, and atherosclerosis in type 1 diabetic mice, we hypothesize that polyphenol-activated SIRT1 acts upstream of AMPK signaling and hepatocellular lipid metabolism. Here we show that polyphenols, including resveratrol and the synthetic polyphenol S17834, increase SIRT1 deacetylase activity, LKB1 phosphorylation at Ser428, and AMPK activity. Polyphenols substantially prevent the impairment in phosphorylation of AMPK and its downstream target, ACC (acetyl-CoA carboxylase), elevation in expression of FAS (fatty acid synthase), and lipid accumulation in human HepG2 hepatocytes exposed to high glucose. These effects of polyphenols are largely abolished by pharmacological and genetic inhibition of SIRT1, suggesting that the stimulation of AMPK and lipid-lowering effect of polyphenols depend on SIRT1 activity. Furthermore, adenoviral overexpression of SIRT1 stimulates the basal AMPK signaling in HepG2 cells and in the mouse liver. AMPK activation by SIRT1 also protects against FAS induction and lipid accumulation caused by high glucose. Moreover, LKB1, but not CaMKKβ, is required for activation of AMPK by polyphenols and SIRT1. These findings suggest that SIRT1 functions as a novel upstream regulator for LKB1/AMPK signaling and plays an essential role in the regulation of hepatocyte lipid metabolism. Targeting SIRT1/LKB1/AMPK signaling by polyphenols may have potential therapeutic implications for dyslipidemia and accelerated atherosclerosis in diabetes and age-related diseases.
732 citations
TL;DR: It is revealed that inactivation of hepatic AMPK is a key event in the pathogenesis of hyperlipidemia in diabetes, point to a novel mechanism of action of polyphenols to lower lipids by activating AMPK, and emphasize a new therapeutic avenue to benefit hyper Lipidemia and atherosclerosis specifically in diabetes.
Abstract: Because polyphenols may have beneficial effects on dyslipidemia, which accelerates atherosclerosis in diabetes, we examined the effect of polyphenols on hepatocellular AMP-activated protein kinase (AMPK) activity and lipid levels, as well as hyperlipidemia and atherogenesis in type 1 diabetic LDL receptor-deficient mice (DMLDLR(-/-)). In HepG2 hepatocytes, polyphenols, including resveratrol (a major polyphenol in red wine), apigenin, and S17834 (a synthetic polyphenol), increased phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase (ACC), and they increased activity of AMPK with 200 times the potency of metformin. The polyphenols also prevented the lipid accumulation that occurred in HepG2 cells exposed to high glucose, and their ability to do so was mimicked and abrogated, respectively, by overexpression of constitutively active and dominant-negative AMPK mutants. Furthermore, treatment of DMLDLR(-/-) mice with S17834 prevented the decrease in AMPK and ACC phosphorylation and the lipid accumulation in the liver, and it also inhibited hyperlipidemia and the acceleration of aortic lesion development. These studies 1) reveal that inactivation of hepatic AMPK is a key event in the pathogenesis of hyperlipidemia in diabetes, 2) point to a novel mechanism of action of polyphenols to lower lipids by activating AMPK, and 3) emphasize a new therapeutic avenue to benefit hyperlipidemia and atherosclerosis specifically in diabetes via activating AMPK.
622 citations
Cites background from "Phosphorylation-activity relationsh..."
...Unless stated otherwise, phosphorylation of ACC was expressed as the ratio of the sum of ACC1 and ACC2 phosphorylation to the level of endogenous AMPK expression....
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...AMPK activation by S17834 (10 mol/l) was further confirmed by enhanced phosphorylation of both ACC1 and ACC2 in HepG2 cells comparable to that of AICAR (1 mmol/l) and metformin (2 mmol/l) (Fig....
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...Our data showing that exposure of HepG2 cells to high glucose inhibits the activity of AMPK 1, the major isoform in liver (14,31), but not AMPK 2 (which is likely due to the low baseline AMPK 2 activity), and decreases phosphorylation of ACC1 and ACC2, resulting in increased ACC activity and elevated hepatic lipids, suggests that inhibition of AMPK 1 activity in hepatocytes is responsible for the high-glucose–induced lipid accumulation....
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...In some cases, ACC1 and ACC2 bands were individually assessed by densitometry....
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...Like the effect of the known AMPK activators, the lipid-lowering effect of polyphenols may be attributable to activation of AMPK and inactivation of ACC1 and ACC2 and consequently their effects to downregulate fatty acid synthesis and upregulate fatty acid oxidation....
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TL;DR: This review will summarize the structural information that is now available for both the BC and CT enzymes, as well as the molecular mechanism of action of potent ACC inhibitors.
Abstract: Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism in most living organisms. Mice deficient in ACC2 have continuous fatty acid oxidation and reduced body fat and body weight, validating this enzyme as a target for drug development against obesity, diabetes and other symptoms of the metabolic syndrome. ACC is a biotin-dependent enzyme and catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA through its two catalytic activities, biotin carboxylase (BC) and carboxyltransferase (CT). ACC is a multi-subunit enzyme in most prokaryotes, whereas it is a large, multi-domain enzyme in most eukaryotes. The activity of the enzyme can be controlled at the transcriptional level as well as by small molecule modulators and covalent modification. This review will summarize the structural information that is now available for both the BC and CT enzymes, as well as the molecular mechanism of action of potent ACC inhibitors. The current intense research on these enzymes could lead to the development of novel therapies against metabolic syndrome and other diseases.
435 citations
TL;DR: Results indicate that ACC beta phosphorylation is especially sensitive to exercise and tightly coupled to AMPK signaling and that AMPK activation does not depend on AMPK kinase activation during exercise.
Abstract: The effect of exercise intensity on skeletal muscle AMP-activated protein kinase (AMPK) signaling and substrate metabolism was examined in eight men cycling for 20 min at each of three sequential intensities: low (40 +/- 2% VO(2) peak), medium (59 +/- 1% VO(2) peak), and high (79 +/- 1% VO(2) peak). Muscle free AMP/ATP ratio only increased at the two higher exercise intensities (P < 0.05). AMPK alpha 1 (1.5-fold) and AMPK alpha 2 (5-fold) activities increased from low to medium intensity, with AMPK alpha 2 activity increasing further from medium to high intensity. The upstream AMPK kinase activity was substantial at rest and only increased 50% with exercise, indicating that, initially, signaling through AMPK did not require AMPK kinase posttranslational modification. Acetyl-CoA carboxylase (ACC)-beta phosphorylation was sensitive to exercise, increasing threefold from rest to low intensity, whereas neuronal NO synthase (nNOS) micro phosphorylation was only observed at the higher exercise intensities. Glucose disappearance (tracer) did not increase from rest to low intensity, but increased sequentially from low to medium to high intensity. Calculated fat oxidation increased from rest to low intensity in parallel with ACC beta phosphorylation, then declined during high intensity. These results indicate that ACC beta phosphorylation is especially sensitive to exercise and tightly coupled to AMPK signaling and that AMPK activation does not depend on AMPK kinase activation during exercise.
349 citations
Cites background from "Phosphorylation-activity relationsh..."
...Similarly, low frequency stimulation in situ and low-intensity running in rats increases skeletal muscle ACC phosphorylation and decreases ACC activity without a detectable increase in stable AMPK activity (5)....
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...A correlation between AMPK activity and Thr-172 phosphorylation was also observed (5)....
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...It has recently been shown that AMPK phosphorylation at Thr-172 increases during in situ stimulations in rat gastrocnemius muscle (5)....
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...Treadmill exercise increases skeletal muscle AMPK activity and ACC phosphorylation and reduces ACC activity and malonyl-CoA content in rats (5,21)....
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...AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC)- (5,20), the enzyme responsible for malonyl-CoA production....
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TL;DR: In conclusion, exercise intensity regulates PGC‐1α mRNA abundance in human skeletal muscle in response to a single bout of exercise, mediated by differential activation of multiple signalling pathways, with ATF‐2 and HDAC phosphorylation proposed as key intensity‐dependent mediators.
Abstract: Skeletal muscle contraction increases intracellular ATP turnover, calcium flux, and mechanical stress, initiating signal transduction pathways that modulate peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha)-dependent transcriptional programmes. The purpose of this study was to determine if the intensity of exercise regulates PGC-1alpha expression in human skeletal muscle, coincident with activation of signalling cascades known to regulate PGC-1alpha transcription. Eight sedentary males expended 400 kcal (1674 kj) during a single bout of cycle ergometer exercise on two separate occasions at either 40% (LO) or 80% (HI) of . Skeletal muscle biopsies from the m. vastus lateralis were taken at rest and at +0, +3 and +19 h after exercise. Energy expenditure during exercise was similar between trials, but the high intensity bout was shorter in duration (LO, 69.9 +/- 4.0 min; HI, 36.0 +/- 2.2 min, P < 0.05) and had a higher rate of glycogen utilization (P < 0.05). PGC-1alpha mRNA abundance increased in an intensity-dependent manner +3 h after exercise (LO, 3.8-fold; HI, 10.2-fold, P < 0.05). AMP-activated protein kinase (AMPK) (2.8-fold, P < 0.05) and calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation (84%, P < 0.05) increased immediately after HI but not LO. p38 mitogen-activated protein kinase (MAPK) phosphorylation increased after both trials (2.0-fold, P < 0.05), but phosphorylation of the downstream transcription factor, activating transcription factor-2 (ATF-2), increased only after HI (2.4-fold, P < 0.05). Cyclic-AMP response element binding protein (CREB) phosphorylation was elevated at +3 h after both trials (80%, P < 0.05) and class IIa histone deacetylase (HDAC) phosphorylation increased only after HI (2.0-fold, P < 0.05). In conclusion, exercise intensity regulates PGC-1alpha mRNA abundance in human skeletal muscle in response to a single bout of exercise. This effect is mediated by differential activation of multiple signalling pathways, with ATF-2 and HDAC phosphorylation proposed as key intensity-dependent mediators.
349 citations
Cites background from "Phosphorylation-activity relationsh..."
...However, AMPK activity may still be increased during low intensity exercise as ACCβ phosphorylation, a sensitive indicator of skeletal muscle AMPK activity (Chen et al. 2003; Park et al. 2002), was increased following both exercise trials (Fig....
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References
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TL;DR: Key developments of the last 20 years that have led to the current understanding of the physiology of the CPT system, the structure of theCPT isoforms, the chromosomal localization of their respective genes, and the identification of mutations in the human population are reviewed.
Abstract: First conceptualized as a mechanism for the mitochondrial transport of long-chain fatty acids in the early 1960s, the carnitine palmitoyltransferase (CPT) system has since come to be recognized as a pivotal component of fuel homeostasis. This is by virtue of the unique sensitivity of the outer membrane CPT I to the simple molecule, malonyl-CoA. In addition, both CPT I and the inner membrane enzyme, CPT II, have proved to be loci of inherited defects, some with disastrous consequences. Early efforts using classical approaches to characterize the CPT proteins in terms of structure/function/regulatory relationships gave rise to confusion and protracted debate. By contrast, recent application of molecular biological tools has brought major enlightenment at an exponential pace. Here we review some key developments of the last 20 years that have led to our current understanding of the physiology of the CPT system, the structure of the CPT isoforms, the chromosomal localization of their respective genes, and the identification of mutations in the human population.
1,560 citations
TL;DR: The primary focus of this lecture will be on the first of these issues, which is the pathophysiological basis for type 2 diabetes and why is their rate of appearance accelerating so rapidly at this particular juncture of human history.
Abstract: Readers of this journal do not need to be reminded of the explosive increase in obesity/type 2 diabetes syndromes and their attendant staggering public health costs that are currently afflicting developed countries. Because of this alarming development and what it portends for the future if left unchecked, the American Diabetes Association, together with its counterparts around the world, as well as the World Health Organization, are faced with the daunting challenge of finding answers to two huge questions. First, what is the pathophysiological basis for these disorders? Second, why is their rate of appearance accelerating so rapidly at this particular juncture of human history, and what, if anything, can we do to intervene? The primary focus of this lecture will be on the first of these issues. I want to emphasize at the outset that I do not plan to dwell exclusively on our own studies in this area. Instead, I shall try to incorporate recent developments from a variety of other laboratories working in the field. Although these will of necessity have to be highly selective, I hope that collectively they will illustrate how we have come to a relatively new and exciting way of thinking about the etiology of type 2 diabetes.
It has been said in literary circles that James Joyce’s Ulysses is probably the most frequently opened and least read book that has ever been published. In a similar vein, I sometimes think that the question of what causes type 2 diabetes might be one of the most frequently asked and least satisfactorily answered in the history of diabetes research. This is not meant to be an arrogant statement, nor indeed does it imply that I have a magical solution to the question at hand. Rather, it simply reflects the fact that at the dawn of …
1,464 citations
TL;DR: The central hypothesis is that the AMP-activated protein kinase cascade appears to be an ancient system which evolved to protect cells against the effects of nutritional or environmental stress, and protects the cell by switching off ATP-consuming pathways and switching on alternative pathways for ATP generation.
Abstract: A single entity, the AMP-activated protein kinase (AMPK), phosphorylates and regulates in vivo hydroxymethylglutaryl-CoA reductase and acetyl-CoA carboxylase (key regulatory enzymes of sterol synthesis and fatty acid synthesis, respectively), and probably many additional targets. The kinase is activated by high AMP and low ATP via a complex mechanism, which involves allosteric regulation, promotion of phosphorylation by an upstream protein kinase (AMPK kinase), and inhibition of dephosphorylation. This protein-kinase cascade represents a sensitive system, which is activated by cellular stresses that deplete ATP, and thus acts like a cellular fuel gauge. Our central hypothesis is that, when it detects a 'low-fuel' situation, it protects the cell by switching off ATP-consuming pathways (e.g. fatty acid synthesis and sterol synthesis) and switching on alternative pathways for ATP generation (e.g. fatty acid oxidation). Native AMP-activated protein kinase is a heterotrimer consisting of a catalytic alpha subunit, and beta and gamma subunits, which are also essential for activity. All three subunits have homologues in budding yeast, which are components of the SNF1 protein-kinase complex. SNF1 is activated by glucose starvation (which in yeast leads to ATP depletion) and genetic studies have shown that it is involved in derepression of glucose-repressed genes. This raises the intriguing possibility that AMPK may regulate gene expression in mammals. AMPK/SNF1 homologues are found in higher plants, and this protein-kinase cascade appears to be an ancient system which evolved to protect cells against the effects of nutritional or environmental stress.
1,310 citations
TL;DR: This finding is consistent with the recent report that the AMP-activated protein kinase kinase can slowly phosphorylate and activate calmodulin-dependentprotein kinase I, at least in vitro.
Abstract: We have developed a sensitive assay for the AMP-activated protein kinase kinase, the upstream component in the AMP-activated protein kinase cascade. Phosphorylation and activation of the downstream kinase by the upstream kinase absolutely requires AMP and is antagonized by high (millimolar) concentrations of ATP. We have purified the upstream kinase >1000-fold from rat liver; a variety of evidence indicates that the catalytic subunit may be a polypeptide of 58 kDa. The physical properties of the downstream and upstream kinases, e.g. catalytic subunit masses (63 versus 58 kDa) and native molecular masses (190 versus 195 kDa), are very similar. However, unlike the downstream kinase, the upstream kinase is not inactivated by protein phosphatases. The upstream kinase phosphorylates the downstream kinase at a single major site on the α subunit, i.e. threonine 172, which lies in the “activation segment” between the DFG and APE motifs. This site aligns with activating phosphorylation sites on many other protein kinases, including Thr177 on calmodulin-dependent protein kinase I. As well as suggesting a mechanism of activation of AMP-activated protein kinase, this finding is consistent with our recent report that the AMP-activated protein kinase kinase can slowly phosphorylate and activate calmodulin-dependent protein kinase I, at least in vitro (Hawley, S. A., Selbert, M. A., Goldstein, E. G., Edelman, A. M., Carling, D., and Hardie, D. G. (1995) J. Biol. Chem. 270, 27186-27191).
1,235 citations
"Phosphorylation-activity relationsh..." refers background in this paper
...Studies have also demonstrated that AMPK can be phosphorylated on threonine 172 of the -subunit, resulting in activation (6, 14, 16, 18, 43)....
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TL;DR: Evidence is provided that decreases in muscle content of malonyl-CoA can increase the rate of fatty acid oxidation, and perfusion with medium containing AICAR was found to activate AMPK in skeletal muscle, inactivate ACC, and decrease malony l-coA.
Abstract: 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) has previously been reported to be taken up into cells and phosphorylated to form ZMP, an analog of 5′-AMP This study was designed to determin
1,012 citations
"Phosphorylation-activity relationsh..." refers methods in this paper
...Hindlimb perfusion studies using 5-aminoimidazole-4carboxamide-1- -D-ribofuranoside to activate AMPK also show an inverse relationship between ACC activity (and consequently malonyl-CoA concentration) and fatty acid oxidation (27, 28)....
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